The process of inflammation is characterized by the rapid influx of a particular granulocyte, the neutrophil, into the affected area as a response to either physical injury or pathogen invasion1,2. Detection of elevated neutrophil levels, therefore, represents a marker for numerous inflammatory events and conditions that can be useful in the diagnosis and subsequent treatment of disease. Three examples of areas in which detection of neutrophils is of clinical value are intra-mammary mastitis in cows and other domesticated herbivores3, in acute respiratory distress syndrome (ARDS) in humans and in equine inflammatory airway disease (EIAD)4.
Traditionally neutrophils in tissue or bodily fluids are detected and/or enumerated by either one of two ways. The first is by using manual microscopic examination of samples under high power following cytological staining (such as hematoxylin/eosin) where they are identified by their multi-lobed nuclei and characteristic cytoplasmic granules. The second uses flow cytometry, where immune cells can be differentiated by their relative sizes in combination with their different light-scattering properties.
While both such methodologies might be readily available to large medical practices associated with hospitals, and to some well-equipped veterinary clinics, in most cases samples must be sent to diagnostic laboratories resulting in delays to patient treatment. In large-animal veterinary practice in particular, where on-site visits are common practice, rapid detection of neutrophil influx is not available because high-power microscopes are bulky and inconvenient, and histology and examination are time-consuming, while flow cytometers are delicate pieces of equipment that are not suitable for routine relocation from site to site. There is, therefore, a strong need for a rapid (5-10 minutes or less), affordable and portable technology to facilitate the on-site detection of elevated neutrophil levels by either veterinary practitioners (and even farmers) or by smaller human medical practices that do not have the resources to purchase, or access to, sophisticated laboratory equipment.
This application describes an invention aimed at addressing this unmet need and that is of particular utility to the dairy industry. More specifically, the invention is particularly useful in providing early diagnosis of mammary gland infection in cattle and other domesticated mammals (for example, sheep and goats).
Mastitis is a ubiquitous disease caused by the invasion of the mammary gland by bacteria and the subsequent resulting inflammatory response. It is one of the most widespread diseases affecting the dairy industry, leading to annual losses of $2 billion in the US alone5,6. Numerous factors contribute to such losses, including: milk production drops; disposal of milk deemed unfit for consumption; cost of drugs, veterinary care and diagnostics; additional labor; and, in rarer cases death of the animal. Losses occur even in the absence of evident disease since milk production drops significantly even the case of subclinical mastitis7-9.
Earlier diagnosis of mastitic infections can significantly reduce their economic burden and increase efficiency in a number of these areas. Not only does early detection lead to earlier treatment and more rapid resolution, with a concomitant decrease in the risks of serious complications and even death, but also to production savings from both increased yields and a decrease in milk withheld from the market due to adulteration with antibiotics. Furthermore, mastitis in dry cows can have serious clinical consequences for the animal and also results in significant decreases in milk production in subsequent lactations. Thus Dry Cow Therapy (DCT), i.e. prophylactic antibiotic infusion of the udder at drying-off in combination with a teat sealant is a common practice10,11 that both increases operating costs due to the price of the antibiotics and risks selection of resistant bacterial strains12,13. The latter concern could lead to eventual regulation of the prophylactic use of these compounds, as has already occurred with feed, and so anything that would encourage their more prudent use is to be encouraged14. In this case, convenient subclinical mastitis tests that could identify infected animals or udder quarters cow-side and so promote more effective selective DCT (sDCT)15-17 would also yield increased efficiencies for dairy farmers while lowering the costs and resistance risks associated with antibiotic-based prophylaxis.
Mastitis is diagnosed by the appearance of the udder (e.g. redness, temperature, swelling, hardness or pain), and/or by changes in milk appearance (e.g. increased wateriness or appearance of clots, pus or flakes). By this point, however, infection has been firmly established, making the condition more challenging and time-consuming to treat. In addition, milk yields begin to decrease substantially before any clinical symptoms present18-22, leading to significant economic losses even in such asymptomatic cases, amounting to $1 billion annually in the U.S. alone23.
Subclinical mastitis can be diagnosed by bacterial culture of aseptically collected milk, though this is both expensive and time consuming. As a result, the prevalence of latent mastitis in both herds and individual animals is most commonly diagnosed using a surrogate marker, the somatic cell count (SCC)24, since it has been demonstrated that elevated counts are prognostic of bacterial infection25 and susceptibility to developing clinical symptoms26.
The utility of SCCs reaches beyond early the treatment of infection, and testing results have additional economic consequences to the farmer. Elevated-SCC milk has a shorter shelf-life and produces lower-quality milk-products27-31. It also produces lower yields of milk-derived foodstuffs such as cheese32. As a result, many milk distributors offer quality premiums for milk low-SCC milk, further inducing farmers to manage subclinical mastitis in their operations33. Our proposed animal-side technology would facilitate earlier detection of mastitis at lower SCCs, making it easier for the dairyman to isolate infected animals and so to capitalize on these incentives.
Furthermore, regulatory authorities set strict limits for maximum allowable counts in milk shipments, with bulk tank SCC (BTSCC) being 750,000 cells/ml in the U.S. and 400,000 in Europe34, resulting in the wastage of shipments that do not meet these criteria.
Thus, regular SCC testing provides an opportunity for dairy farmers to increase the efficiency of their operations as well as to enhance the well-being of their cattle, and so the development of technologies aimed at improving the sensitivity, reducing the cost, and diminishing the inconvenience of monitoring their herds would be welcomed.
Control of mastitis depends on both a herd and animal-level approach to conducting SCCs35. BTSCCs serve to monitor the mastitic state of the entire herd (as well as a quality control point for the milk itself), and increases in BTSCCs can serve as a signal of underlying subclinical mastitis issues. However, due to the dilution of milk from high SCC cows with that of the entire herd, BTSCCs lack sensitivity and so provide a “delayed” warning of a growing problem. As a result, testing of individual animals sampled from the herd is also conducted. This not only facilitates early identification of infected animals but also identifies those contributing to an elevated BTSCC so that they can be isolated and treated.
While alternative approaches, such as measuring the changes in electrical conductivity of milk and measuring the activity of various enzymes, have been proposed or are in use, none possess either the specificity or sensitivity of the SCC36.
Numerous methods exist for subclinical mastitis diagnosis based on elevated cellular levels in milk. These can be divided into three broad classes: laboratory, on-farm and cow-side methods.
Laboratory methods utilize sophisticated flow cytometry either without or in conjunction with fluorescent staining. Such methods rely on shipment of samples from farms for analysis, which leads to delays between sample collection and receipt of results. A number of commercial devices are available for use in such diagnostic laboratories, including the Fossomatic (Foss) and the Delta SomaScope (Perten Instruments). Farmers using such services typically test once a month and therefore can only track the status of their animals with a temporal resolution of 30 days.
On-farm devices avoid the inconvenience of shipping samples and the associated time delays, and a number of models are available to veterinarians and farmers based on the imaging and computational enumeration of fluorescently labeled cells. These include the Lactoscan SCC (Milkotronic Ltd.), the Nucleocounter SCC (Chemometec) and the QScout (Advanced Animal Diagnostics). These devices are intended to be used in a dedicated laboratory space on the farm, but do not require highly trained operators and can provide same-day test results to the farmer.
Animal-side devices are intended to be used outside of the laboratory setting and provide the benefit of providing SCC information animal-side so that treatment/livestock management decisions can be made immediately. Some of these tests are relatively sophisticated and also rely on the electronic enumeration of fluorescently stained cells, for example, the Cell Counter ICC (DeLaval Inc.) and the Dairy Quality SCC (Dairy Quality). However, older and cruder tests are also available that based on the gelation of somatic cell DNA upon lysis with detergent. These include the California Mastitis Test (CMT)37,38, where gelation is semi-quantitative and subject to user visual-analog scoring of the degree of milk gelation, and the Wisconsin Mastitis Test (WMT)39, where the gel is quantified using a graduated scale. These tests are available from numerous vendors, including the Somaticell SCC test (IDEXX).
Two other differentiating factors between these tests are the ability to count all four udder quarters simultaneously, and the ability to differentiate between different milk leukocytes. The former provides enhanced sensitivity not only because the cell count of an affected quarter is not diluted by their non-infected counterparts40-43, but also because the counts of uninfected quarters can serve as internal negative controls since SCCs vary dramatically from animal to animal and even during lactation44. The latter also provides increases sensitivity because the activation and influx of neutrophils occurs very soon after microbial invasion (see below)45-48. Numerous researchers have suggested that such differential somatic cell counts (DSCCs) provide an earlier indication of subclinical mastitis than SCCs alone, and the focus of their work has been the detection of elevated levels of neutrophils49-55.
Of the currently available tests, the CMT provides the ability of test all four udder quarters simultaneously, while the Fossamatic provides the capability of conducting DSCCs and yet these options lie on opposite ends of the portability spectrum. The only user-friendly device currently capable of conducting DSCCs on-farm is the QScout, though it is too bulky to be considered as a truly portable unit and is designed to be used in a dedicated laboratory space.
Furthermore, cost is also a consideration. While the CMT is relatively affordable (inexpensive reagents coupled with reusable hardware) it lacks the sensitivity of direct cell counting and is prone to user subjectivity. SCCs from commercial reference laboratories are relatively inexpensive, but their cost mounts when analyzing four quarters and there is a delay in obtaining results. Animal-side devices use disposal plastic cassettes that cost several dollars per test and cannot analyze all four quarters simultaneously. Finally, the QScout, which can be used by the average farmer, albeit not animal-side, does analyze all four quarters but also uses disposable cassettes which cost approximately $5 per test while the QScout itself costs almost $20,000.
Thus, veterinarians' and farmers' current options regarding SCCs are a trade-off between cost, the convenience of animal-side tests and the sensitivity afforded by laboratory-based methods. In addition to diagnosing other inflammatory conditions in both humans and other animals, this invention addresses the unmet need for a portable and affordable DSCC test that can be used animal-side to provide early diagnosis of subclinical mastitis.